WO2020082950A1 - 鞘氨醇激酶1及其融合蛋白及其用途 - Google Patents

鞘氨醇激酶1及其融合蛋白及其用途 Download PDF

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WO2020082950A1
WO2020082950A1 PCT/CN2019/107091 CN2019107091W WO2020082950A1 WO 2020082950 A1 WO2020082950 A1 WO 2020082950A1 CN 2019107091 W CN2019107091 W CN 2019107091W WO 2020082950 A1 WO2020082950 A1 WO 2020082950A1
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acid sequence
protein
expression construct
vector
amino acid
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French (fr)
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段海峰
解晶
胡显文
弓景波
薛冰华
张群伟
肖秀孝
崔美兰
庞如梦
王瑞
于婷婷
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Beijing Shuangyin Biotechnology Co Ltd
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Priority to US17/286,445 priority patent/US20210388326A1/en
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01091—Sphinganine kinase (2.7.1.91)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to the use of sphingosine kinase 1, more specifically, the present invention relates to a fusion protein of sphingosine kinase 1, in particular to a fusion protein containing sphingosine kinase 1 and FC sequences and use.
  • T2DM type 2 diabetes
  • the drugs currently used to treat diabetes mainly include insulin and oral hypoglycemic drugs such as metformin, but the shortcomings of these drugs are that they easily cause hypoglycemia and have no obvious effect on the control of the patient's weight.
  • Another class of drugs are GLP-1 receptor agonist drugs, such as Liraglutide from Novo Nordisk and Dularutide from Eli Lilly.
  • Such drugs can also reduce weight while controlling blood sugar, but it is mainly achieved by suppressing the patient's appetite and controlling the patient's food intake, which greatly reduces the patient's quality of life.
  • SphK1 sphingosine kinase 1
  • S1P sphingosine-1-phosphate
  • SphK1 catalyzes the production of S1P from sphingosine, a metabolite of ceramide.
  • S1P can regulate cell processes such as cell growth, apoptosis, differentiation and hematopoiesis after binding to receptors.
  • the SphK1 / S1P signaling pathway is involved in various biological processes and diseases, including tumorigenesis and diabetes.
  • the prior art shows that SphK1 can be secreted out of the cell, but its role in the extracellular environment and the presence of extracellular receptors are unclear (Venkataraman K, et al. Biochemical Journal, 2006, 397 (3): 46 1-71 .).
  • mice lacking SphK1 can promote pancreatic cell apoptosis under a high-sugar and high-fat diet, thereby inducing the formation of diabetes (Qi Y, et al. Faseb Journal, 2013, 27 (10): 4294-4304).
  • diabetic mice after injection of adenovirus carrying the human SphK1 gene, showed reduced blood glucose and blood lipid levels compared to control mice.
  • the research on SphK1 is mainly based on its role in cells, so the drugs developed with this protein as the target mainly use its antibodies or antagonists, and the protein is not directly made into drugs for treatment.
  • the other is to use the virus as a carrier to pour the SPHK1 gene into cells for gene therapy, such as gene therapy using adenovirus as a carrier.
  • this method is easy to develop drug resistance in the body, and the treatment of metabolic diseases such as diabetes requires long-term Medication, which limits the use of this method. Therefore, drug development based on human SPHK1 gene has broad prospects.
  • the object of the present invention is to provide a use of sphingosine kinase 1 against the deficiencies of the prior art.
  • the inventor unexpectedly discovered that SphK1 is directly made into protein drugs, so that it can function outside the cell without entering the cell, and has a significant effect of lowering blood sugar and weight. Therefore, the present invention provides a use of sphingosine kinase 1 in the preparation of a medicament for preventing and / or treating obesity, hyperlipidemia, or diabetes.
  • the present invention also provides a class of protein drugs, the drug comprises sphingosine kinase 1 (SPHK1), and the present invention also provides a preparation method and use of the protein drugs.
  • the protein drugs provided by the present invention can significantly reduce blood sugar, blood lipids, body weight and improve fat metabolism.
  • the present invention provides a use of sphingosine kinase 1 or an amino acid sequence having its activity in the preparation of protein drugs for preventing and / or treating obesity, hyperlipidemia or diabetes.
  • the sphingosine kinase 1 or the amino acid sequence having its activity comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the present invention provides a protein drug, the protein drug comprising sphingosine kinase 1 or an amino acid sequence having its activity;
  • the proteinaceous drug is a fusion protein containing sphingosine kinase 1 or an amino acid sequence having activity; more preferably, the fusion protein comprises sphingosine kinase 1 (SPHK1) or an amino acid sequence having activity , FC sequence and connection sequence;
  • SPHK1 sphingosine kinase 1
  • the FC sequence is selected from the amino acid sequence of human or animal immunoglobulin and its subtypes and variants, or the amino acid sequence of human or animal albumin and its variants;
  • connection sequence is (GGGGS) n, where n is an integer of 0-5; preferably, n is 3;
  • the human or animal immunoglobulin is selected from IgG4FC fragments; more preferably, the human or animal immunoglobulin is selected from the amino acid sequence shown in SEQ ID NO: 12;
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the fusion protein is modified with polyethylene glycol; preferably, the average molecular weight of the polyethylene glycol is 5-50KD; more preferably, 20-45KD; preferably, the poly Ethylene glycol is a linear or branched polyethylene glycol.
  • the present invention provides a coding gene, wherein the coding gene contains the coding nucleotide sequence of the above-mentioned protein drugs; preferably, the coding nucleotide sequence is shown in SEQ ID NO: 3.
  • the present invention provides an expression construct, the expression construct containing the nucleotide sequence encoding the above protein drugs; preferably, the encoding nucleotide sequence is shown in SEQ ID NO: 3.
  • the expression construct is a prokaryotic expression construct; more preferably, the prokaryotic expression construct is a pET vector series;
  • the expression construct is a eukaryotic expression construct; preferably, the eukaryotic expression construct is a plasmid DNA vector, preferably a pVAX1 vector and a pSV1.0 vector; a recombinant viral vector, preferably a recombinant vaccinia virus vector, a recombinant adenovirus Vector or recombinant adeno-associated viral vector; or retroviral vector, preferably HIV viral vector, or lentiviral vector.
  • the eukaryotic expression construct is a plasmid DNA vector, preferably a pVAX1 vector and a pSV1.0 vector
  • a recombinant viral vector preferably a recombinant vaccinia virus vector, a recombinant adenovirus Vector or recombinant adeno-associated viral vector
  • retroviral vector preferably HIV viral vector, or lentiviral vector.
  • the present invention provides a host cell, the host cell comprising the above expression construct
  • the host cell is a prokaryotic cell, preferably a bacterial cell; or when the expression construct is a eukaryotic expression construct, the host cell is true Nuclear organism cells, preferably mammalian cells, more preferably CHO cells.
  • the present invention provides a method for preparing a protein drug, the method including the step of cloning the nucleotide sequence of the protein drug into an expression vector.
  • the preparation method includes the following steps:
  • step 2) The expression vector of step 2) is used to transfect or transform a host cell, and the nucleic acid sequence is expressed in the host cell;
  • the host cell is a CHO-S cell.
  • the invention also provides the application of the above-mentioned protein drugs, coding genes, expression constructs, and the host cells in the preparation of pharmaceutical compositions for preventing and / or treating obesity, hyperlipidemia, or diabetes.
  • the present invention Compared with the prior art, the present invention has the following advantages: The present invention finds that sphingosine kinase 1 and its fusion protein have significant blood sugar lowering and weight loss effects, and can be used to prepare obesity and other metabolic diseases control and diabetes protein Drugs.
  • Figure 1 is a schematic diagram of the vector construction of pCDH-SPHK1-L-Fc of the present invention
  • Figure 2 is the expression of protein SPHK1-Fc by Western blotting, where a is the expression of protein in the supernatant after lentivirus infection of cells ; "Blank” is the supernatant of virus-infected cells, detected with human IgG4Fc specific antibody; b is the purified SDS-PAGE electrophoresis diagram
  • Figure 3 shows the SPHK1 protein of the present invention and its fusion protein SPHK1-Fc to type II The effect of fasting blood glucose in diabetic model mice; the control is the saline group; * represents a significant difference compared to the control (p value ⁇ 0.05);
  • Figure 4 shows the SPHK1 protein and its fusion protein SPHK1-Fc of the present invention after 2 weeks of treatment Effect of body weight on type II diabetes model mice; control is saline group; * represents significant difference compared to control (p value ⁇ 0.05
  • the reagents used in the following examples are analytical grade reagents, and are commercially available from regular channels.
  • the sphingosine kinase 1 or the amino acid sequence having its activity includes SEQ ID NO: 1;
  • the coding nucleotide sequence is shown in SEQ ID NO: 5;
  • the amino acid sequence of the fusion protein SPHK1-Fc is shown in SEQ ID NO: 2, and its nucleotide sequence is shown in SEQ ID NO: 3. Its N segment to C segment are SPHK1, connecting sequence L and Fc in sequence;
  • amino acid sequence of Fc is shown in SEQ ID NO: 12:
  • amino acid sequence of the fusion protein SPHK1-Fc is shown in SEQ ID NO: 2, where the bold italic part is the amino acid sequence of the linking sequence, and the underlined part is the amino acid sequence of Fc:
  • the nucleotide sequence is shown in SEQ ID NO: 3, where the italic part is the nucleotide sequence of the linking sequence, and the underlined part is the Fc nucleotide sequence:
  • the signal peptide sequence is the IL-2 signal peptide sequence (SP, the amino acid sequence is shown in SEQ ID NO: 4; SEQ ID NO: 4MYRMQLLSCIALSLALVTNS) primers are designed based on the IL-2 signal peptide, including armSP-F (upstream primer) (SEQ ID NO: 6: CTCCATAGAAGATTCTAGAGCTAGGGATCCGCCACCATGTACAGGATGCAACTCCTG) and armSP-R (downstream primer) (SEQ ID NO: 7: GGGCCGCCCGCTGGGTCCATCGAATTCGTGACAAGTGCAAG), using the plasmid pFUSE-hIgG4-Fc2 (purchased from Dakco PCR Amplification Technology Co., Ltd.) as a template (116bp); design primers based on the nucleotide sequence of SPHK1 such as SEQ ID NO: 5, including SPHK1-F (upstream primer) (SEQ ID NO: 8: ATGGACCCAGCGGGCGGCC) and
  • Annealing temperature of different fragments amplified by PCR is Tm value of primer -3 °C; # PCR extension time of different fragments is 1kb / min.
  • the resulting product was subjected to 1% agarose gel electrophoresis, and gel recovery purification (the gel recovery kit was purchased from Tiangen Company) each PCR product sp, SPHK1, Fc.
  • the plasmid pCDH-CMV (purchased from Addgene) was double digested with BamHI and SalI, and the digested product was excised and recovered by gel, and the above purified PCR products sp, SPHK1, and Fc were cloned seamlessly (Streamless Assembly Cloning Kit, Purchased from Clone Smarter technologies) connection.
  • the ligation product was transformed into DH5 ⁇ competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.).
  • For the transformation method please refer to the instructions of competent cells.
  • the transformed bacterial solution was spread on an LB plate containing 100 ⁇ g / mL ampicillin and incubated overnight at 37 ° C.
  • Inoculate 293T cells (Lab217 Embryo Engineering Laboratory of Northeast Agricultural University) with a cell confluence of over 90% into a 150mm petri dish, 1.2 x 10 7 cells per dish, add 20ml of DMEM medium containing 10% FBS, 37 Cultivation at 5% CO 2 saturation humidity. 2h before transfection, the original medium was discarded and replaced with 18ml of serum-free DMEM medium.
  • the supernatant was collected and stored at 4 ° C, and a new 20ml medium was added. After 24 hours of continuous culture, the supernatant was collected again. The two collected supernatants were centrifuged at 4 ° C and 3500 rpm for 15 min, and the pellet was discarded. The supernatant was centrifuged and concentrated with an Amicon Ultra-15 ultrafiltration tube (10KD, purchased from Millipore) to obtain lentiviral particles carrying SPHK1-Fc respectively. . After the virus titer determination, the virus particles were diluted to 1 ⁇ 10 8 TU / ml, the virus was divided and stored at -80 ° C.
  • the suspended FreeStyle CHO-S cells (purchased from Thermo Scientific) were seeded at 2 ⁇ 10 5 cells / mL in 30 mL CD-SFM medium (CD FortiCHO medium + 8 mM glutamine + 1 ⁇ HT supplement, all purchased from Thermo scientific company) in a 125 mL shake flask (purchased from Corning), 120 rpm, 8% CO 2 , 37 ° C. to logarithmic growth phase, the cells were diluted with CD-SFM medium to 4 ⁇ 10 4 cells / mL cells Suspension.
  • CD-SFM medium CD FortiCHO medium + 8 mM glutamine + 1 ⁇ HT supplement, all purchased from Thermo scientific company
  • the purified sample was measured for protein concentration using BCA protein quantification kit (purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.). According to the quantitative results, 10 ⁇ g of protein was taken and subjected to SDS-PAGE electrophoresis with 10% gel, stained and developed with a rapid protein staining kit (purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.), and the results were scanned and stored. As shown in FIG. 2 (b), the size of the main protein obtained after purification is consistent with the result of immunoblotting in Example 3.1.
  • mice Eighteen 4-8 week old male type 2 diabetes model rats BKS.Cg-Dock7m + / + Leprdb / Nju (purchased from the Institute of Animal Modeling, Nanjing University) were divided into three groups based on body weight and fasting blood glucose: control group (control, physiological Saline), administration group 1 (SPHK1-Fc protein) and administration group 2 (SPHK1 protein, purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Catalog No. 15679-HNCB), 6 mice per group.
  • the administration method of each group is subcutaneous injection, and the dosage is 2mg / kg.
  • the control group and the administration group 1 were administered twice a week; the administration group 2 was administered once a day.
  • Glucose tolerance was measured after 2 weeks of treatment: fasting was measured for 12 hours the night before, glucose was injected intraperitoneally at a dose of 1 g glucose / kg, and blood glucose of mice was measured at 0, 30, 60 and 120 min.
  • the results in Figure 5 show that the blood glucose levels of the two administration groups are lower than those of the control group at each test point, and there is no significant difference in glucose tolerance between the two administration groups, indicating that the mice are tolerant to glucose after administration Sex has a significant improvement.

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PCT/CN2019/107091 2018-10-21 2019-09-20 鞘氨醇激酶1及其融合蛋白及其用途 Ceased WO2020082950A1 (zh)

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US17/286,445 US20210388326A1 (en) 2018-10-21 2019-09-20 Sphingosine kinase 1 and fusion protein comprising the same and use thereof

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